Synaptotagmins are multidomain proteins belonging to a large gene family in animals, which are exclusively or predominantly expressed in neuronal cells. They are proposed to be the Ca2+ sensors that trigger rapid and synchronous synaptic vesicle exocytosis to release neurotransmitter, and subsequent endocytosis to recapture membrane. However, the functions of synaptotgamins in non-neural cells have not been well characterized. Plant virus cell-to-cell spread through the barrier of the plant cell wall is mediated by virus-encoded movement proteins (MPs). Recently, the investigation of this process lead to the unexpected discovery of a five-gene family of synaptotagmins (AtSYTA through AtSYTE) in the model plant Arabidopsis thaliana. Our lab has shown that: (1) SYTA interacts with, and regulates the cell-to-cell trafficking of, the distinct movement proteins (MPs) encoded by a Begomovirus and a Tobamovirus;and (2) SYTA regulates the formation of early endosomes at the plasma membrane in plant cells. We propose that SYTA regulates plant virus cell-to-cell movement, and plant cell macromolecular trafficking, through, plasmodesmata via an endocytic recapture pathway. Our long term goal is to understand the roles of Syts in plant cell function and plant development, and to understand how viruses usurp the endocytic machinery to exit from an infected cell. The proposed research will focus on characterizing the interactions of SYTA with a variety of diverse virus movement proteins, and the functions of SYTC in virus movement and plant development. Two specific aims are proposed: (1) To investigate the roles of SYTA in virus movement using cell trafficking, infectivity, in vitro protein binding, and live, cell imaging assays;and (2) To determine if SYTA and SYTC have overlapping functions in virus movement and in plant development by characterizing sytc and syta/sytc mutant Arabidopsis lines, determining developmental pattern of SYTC expression, and characterizing the interaction of SYTC with viral MPs. This research will utilize a newly developed biolistic assay to transiently express proteins in Arabidopsis leaf cells and multiphoton confocal microscopy to examine protein interactions in intact leaves in real time. The proposed research can lead to rational approaches to engineer virus-resistant plants, thus improving agricultural production and human nutrition. Beyond this, these studies can inform research in invertebrate and mammalian systems on the functions of Syts in non-neural cells by providing new insights relevant to understanding the roles of animal Syts and vesicle trafficking in development, and how animal and human viruses such as retroviruses like HIV may take advantage of vesicle trafficking for virus assembly and exit.